Directed antisense libraries

ABSTRACT

A method for making a directed antisense library against a target transcript is described. A cDNA of the target transcript is cloned in an appropriate cloning vector. Next, a plurality of deletion derivatives of the cloned cDNA is prepared such that the deletions serially extend into the cDNA from one end thereof. The resulting deletion library is then treated such that cDNA is removed from the other end of each cDNA insert, thus obtaining a fragment library having fragments of a selected size. A catalytic core is then inserted into each fragment of the fragment library, resulting in the directed antisense library. An illustrative antisense gene in the hammerhead ribozyme catalytic core. Plasmids for making the antisense library, plasmids and methods for making the fragment library, and a method for identifying target sites for antisense-mediated gene inhibition are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is an application under 35 U.S.C. §371 of InternationalApplication No. PCT/US99/06742, which has an international filing dateof Mar. 28, 1999, which claims the benefit of U.S. ProvisionalApplication No. 60/079,792, filed Mar. 28, 1998, and U.S. ProvisionalApplication No. 60/107,504, filed Nov. 6, 1998.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.1R03RR08849 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to antisense agents. More particularly, theinvention relates to compositions and methods for generation of directedantisense libraries and methods of use thereof wherein the antisenseagents in the libraries can potentially bind to every binding site on aselected RNA transcript.

Antisense RNA, DNA, and ribozymes have been widely studied as researchtools and potential therapeutic agents for inhibiting the expression ofspecific genes. These agents operate by binding to a complementaryregion on an RNA transcript produced from the gene of interest. Onbinding, the antisense agent can prevent expression of the RNA, and thiscan occur through a variety of different mechanisms. There are manysites on any given RNA for targeted inhibition by an antisense molecule.For a typical RNA transcript of 2000 nucleotides, just under 2000 targetsites are available. Examination of a few to tens of randomly chosentarget sites reveals a great variability in activity. Clearly, not alltarget sites are equivalent in their ability to permit antisensemediated inhibition. Consequently, identification of effective targetsites on the RNA transcript for interaction with the antisense moleculeis imperative for successful application of antisense technology.Methods currently available for this purpose include the use of computeralgorithms to predict target accessibility based on the predictedsecondary structure of the mRNA, the use of randomized oligonucleotideand ribozyme libraries in cell free systems, and the examination of afew to tens of antisense oligonucleotides, targeted to arbitrarilychosen sites, in cell culture assays. These approaches have met withlimited success.

To identify the most effective target site(s), the following conditionsshould be met. First, all possible sites on the target RNA should beevaluated Second, evaluation should be carried out in the normalcellular milieu. This insures that the target is in its naturalstructure, associated with its normal complement of cellular factors.Additionally, the antisense agent has the opportunity to act onalternate structures that may arise as a result of the many RNAprocessing reactions.

To evaluate all target sites, antisense libraries must be used. Theselibraries should contain antisense molecules targeted to every site. Oneapproach is the use of completely randomized DNA, RNA, or ribozymelibraries. The use of completely randomized libraries suffers from twomajor disadvantages. First, while such libraries may contain antisensemolecules directed at all sites on the target RNA, they also containantisense molecules directed at all sites of all potential RNAtranscripts produced by the cell. Therefore, these random librariespotentially have the capability to inhibit expression of every gene inthe cell. Because of this, random libraries are limited to in vitro usein cell free assays. Second, the complexity of these libraries isenormous. For example, a random library that uses 14 nucleotides torecognize its target must contain at least 2.6×10⁸ (i.e., 4¹⁴) differentmembers. Realistically, the size of the library must be at least 10- to100-fold greater in size to insure representation of all sequences. Theproduction and screening of such large libraries is likely beyondcurrent capabilities.

Herein there is described a new method for identifying optimal antisensetarget sites against any desired RNA transcript This is a directedlibrary approach. In other words, this approach uses an antisenselibrary that targets every site on any selected RNA and only sitespresent on the selected RNA. This library, therefore, does not inhibitother non-target RNA transcripts. This approach is also an improvementover known methods because it uses relatively small libraries. Forexample, a library targeting an RNA transcript of 2000 nucleotides, andusing 14 nucleotides to recognize its target, theoretically needs 1986members. In practice, the library would need to be 10- to 50-times thissize. At 50 times, or 99,300 members, this is still a relatively smalllibrary. These directed libraries can be used in both in vitro and invivo assays for the detection of effective target sites for antisensemediated gene inhibition.

In view of the foregoing, it will be appreciated that a method forgenerating directed antisense libraries would be a significantadvancement in the art. Herein is described a method for examining theentire length of any RNA transcript for sites that are accessible toantisense agents. This approach allows for the localization of the mosteffective sites for targeting with antisense agents.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide a simple andinexpensive method for producing directed antisense libraries againstany selected RNA transcript.

It is also an object of the invention to provide a method of producingdirected antisense libraries wherein such libraries contain antisenseagents directed against all targets spanning the entire selected RNAtranscript.

It is another object of the invention to provide a method of usingdirected antisense libraries for locating efficient target sites on theselected RNA transcript.

It is still another object of the invention to provide compositions foruse in constructing directed antisense libraries.

It is yet another object to provide a method for making fragmentlibraries of a selected size of DNA fragment inserted in a cloningvector.

These and other objects can be addressed by providing a method forgenerating an antisense library targeted to a selected RNA transcriptcomprising:

(a) preparing a double-stranded cDNA, comprising a first end, a secondend, and a central site thereof, from the selected RNA transcript andcloning the cDNA in a cloning vector comprising a promoter configuredsuch that an antisense transcript of the cDNA is synthesized upontranscription mediated by the promoter, resulting in a cloned cDNA;

(b) creating a plurality of deletion derivatives of the cloned cDNAwherein each of the plurality of deletion derivatives has a deletionextending from the first end into the cloned cDNA such that theplurality of deletion derivatives comprises a deletion librarycomprising deletions extend serially into the cDNA;

(c) reducing the size of the cDNA contained in the deletion library to apreselected size by removing a portion of the cDNA from the second endthereof to result in a fragment library;

(d) inserting an antisense gene DNA into the central site of the cDNA inthe fragment library, thereby obtaining the antisense library.

Preferred cloning vectors comprise multi-cloning sequences comprisingSEQ ID NO:1 and a combination of SEQ ID NO:2 and SEQ ID NO:3. In apreferred embodiment of the invention, the deletion derivatives arecreated with exonuclease III resection of the cloned cDNA. The size ofthe cDNA contained in the deletion library is preferably reduced to apreselected size by digesting the deletion library with a type IISrestriction endonuclease. Further, inserting the antisense gene DNA intothe central site of the cDNA in the fragment library preferablycomprises digesting the fragment library with a type IIS restrictionendonuclease, thereby creating the central site, and ligating theantisense gene DNA at the central site. A preferred antisense genecomprises a ribozyme catalytic core, more preferably, a hammerheadribozyme catalytic core.

Another aspect of the invention relates to a method for generating alibrary of DNA fragments of a selected size wherein the fragmentscollectively span all possible sites of the selected size in a sourceDNA comprising a first end, a second end, and a central site thereof,comprising:

(a) cloning the source DNA in a cloning vector,

(b) creating a plurality of deletion derivatives of the cloned sourceDNA wherein each of the plurality of deletion derivatives has a deletionextending from the first end into the cloned DNA such that the pluralityof deletion derivatives comprises a deletion library comprisingdeletions extend serially into the cloned DNA; and

(c) reducing the size of the DNA contained in the deletion library to apreselected size by removing a portion of the DNA from the second endthereof to result in the library of fragments.

Still another aspect of the invention relates to a method foridentifying target sites for antisense-mediated inhibition of a selectedgene comprising:

(a) constructing a directed antisense library targeted at the selectedgene wherein the library is contained in a cloning vector having apromoter configured for transcribing antisense transcripts from thedirected antisense library in suitable cells wherein the selected geneis expressed as a target transcript;

(b) transforming a plurality of the suitable cells such that each of theplurality of suitable cells transcribes an antisense transcript that hasaccess to the target transcript for potential inactivation thereof;

(c) identifying a cell wherein an antisense transcript inactivates thetarget transcript; and

(d) analyzing the antisense transcript that inactivates the targettranscript and determining a target site on the antisense transcriptthat is associated with inactivation of the target transcript.

Yet another aspect of the invention relates to a method for identifyingtarget sites for antisense-mediated inhibition of a selected genecomprising:

(a) constructing a directed antisense library targeted at the selectedgene wherein the library is contained in a cloning vector having apromoter configured for transcribing antisense transcripts from thedirected antisense library in vitro;

(b) transcribing antisense transcripts from the directed antisenselibrary in vitro;

(c) incubating the antisense transcripts with a lysate from a cellcontaining target transcripts transcribed from the selected gene suchthat antisense transcripts targeted to the target transcripts bind tosuch target transcripts; and

(d) analyzing the antisense transcripts that bind the target transcriptand determining a target site on the antisense transcript that isassociated with binding of the target transcript.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B show illustrative multi-cloning sequences (MCS's; SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3) according to an aspect of the presentinvention.

FIG. 2 summarizes an illustrative method for making a DNA fragmentlibrary (SEQ ID NO:39) containing 14 bp fragments using the MCS of FIG.1A (SEQ ID NO:1) according to the present invention; also shown areintermediates in the construction of the DNA fragment library (SEQ IDNO:35 through SEQ ID NO:38).

FIG. 3 shows a schematic representation of a hammerhead ribozyme (SEQ IDNO:40) bound to a target substrate (SEQ ID NO:41), wherein X representsA, C, or U; the hammerhead ribozyme comprises a catalytic core thatcleaves the substrate at the cleavage site indicated by the arrow and arecognition domain for binding to the substrate by base pairing.

FIG. 4 summarizes an illustrative method for making a hammerheadribozyme library (SEQ ID NO:48) from an antisense RNA library (SEQ IDNO:39) according to the present invention; also shown are intermediatesin the construction of the hammerhead ribozyme library (SEQ ID NO:42through SEQ ID NO:47).

FIG. 5A shows an illustrative method for inserting a selected cassetteat an end of a deletion fragment (SEQ ID NO:49) in a deletion fragmentlibrary (SEQ ID NO:39) according to the present invention.

FIG. 5B shows an illustrative method for inserting a selected cassettein a MCS (SEQ ID NO:50) prior to preparation of a deletion fragmentlibrary according to the present invention.

FIG. 6A shows a map of expression vector pBK, which is suitable for usein identifying antisense targets in mammalian cells according to thepresent invention.

FIG. 6B shows base pairing of nucleotides in a multi-cloning sequenceflanked by cis-acting ribozymes (CAR's) (SEQ ID NO:18).

FIG. 7A shows a map of vector pASlib according to the present invention,including sequences of relevant MCS regions (SEQ ID NO:2, SEQ ID NO:3).

FIG. 7B shows a map of vector pShuttle according to the presentinvention.

FIG. 7C shows a map of the MCS of pShuttle according to the presentinvention.

FIG. 8 shows a histogram of the distribution of 56 target sites in anillustrative antisense library according to the present invention.

DETAILED DESCRIPTION

Before the present compositions and methods for generating directedantisense libraries and methods of use thereof are disclosed anddescribed, it is to be understood that this invention is not limited tothe particular configurations, process steps, and materials disclosedherein as such configurations, process steps, and materials may varysomewhat. It is also to be understood that the terminology employedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims and equivalentsthereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outherein.

As used herein, “antisense agent” and similar terms mean antisense RNA,antisense DNA, and ribozymes.

As used herein, “comprising,” “including,” “containing,” “characterizedby,” and grammatical equivalents thereof are inclusive or open-endedterms that do not exclude additional, unrecited elements or methodsteps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

As used herein, “consisting of” and grammatical equivalents thereofexclude any element, step, or ingredient not specified in the claim.

As used herein, “consisting essentially of” and grammatical equivalentsthereof limit the scope of a claim to the specified materials or stepsand those that do not materially affect the basic and novelcharacteristic or characteristics of the claimed invention.

Construction of Directed Antisense Libraries

The present invention includes a procedure that allows construction ofdirected antisense libraries of a variety of types. This requires theuse of specially designed bacterial and/or mammalian plasmid vectors.Most importantly, these vectors possess a specially designedmulti-cloning sequence (MCS). This approach is not restricted to asingle MCS, as many can be designed that allow the procedure to beperformed. Two illustrative MCS's are shown in FIGS. 1A (SEQ ID NO:1)and 1B (SEQ ID NO:2 and SEQ ID NO:3). These simply illustrate twopossible multi-cloning sequences that could be used for this method.While some of the same restriction enzyme sites are used in both ofthese MCS's, such particular sites are not necessarily the only sitesthat could be used. Many other restriction enzyme sites could substitutefor any of the restriction sites, allowing the same procedure to beperformed.

The procedure uses a special multi-cloning sequence and a series ofenzymatic manipulations to produce DNA fragment libraries directedagainst any desired gene of interest. The fragment libraries contain alloverlapping fragments spanning the entire length of the gene of interestTranscription in vitro or in vivo of the DNA fragment allows theproduction of an antisense RNA targeted to the site on the RNAtranscript that is encoded by the DNA fragment. Transcription of theentire DNA fragment library produces all antisense RNA moleculestargeting all positions on the RNA target. Expression of the library inmammalian cells allows identification of effective target sites forantisense-mediated gene inhibition.

The procedure is illustrated in FIG. 2 using the MCS shown in FIG. 1A.Beginning with the MCS 10 in a suitable circular plasmid vector(described in more detail below), a blunt-ended DNA fragment encodingthe gene of interest 14 is ligated into the EcoRV-digested MCS (FIG. 2,step a). Since the gene can be inserted in one of two orientations, aclone is selected, according to methods well known in the in art such asnucleotide sequencing or restriction mapping, wherein the gene insert issuitably oriented. The orientation will depend on the placement of atranscriptional promoter adjacent to the MCS. The orientation of theinsert will be chosen such that the antisense strand of the insert willbe transcribed by the adjacent promoter. Next, a deletion library isprepared. The plasmid containing the gene of interest is digested withboth PmeI and BbeI (FIG. 2, step b). The BbeI terminus is protected fromexonuclease III digestion because of its 3′ overhang 18, while the PmeIterminus 22 is a suitable substrate therefor. The digested plasmid isthen treated with exonuclease III and aliquots are removed over timeinto a stop mixture (FIG. 2, step c). The time points are chosen suchthat deletions are generated after every nucleotide across the entiregene. After exonuclease III digestion, the combined aliquots are treatedwith mung bean nuclease to remove the resulting 5′ overhang (FIG. 2,step c). The termini are then polished with T4 DNA polymerase (FIG. 2,step d) and the plasmid is re-circularized with T4 DNA ligase to producethe deletion library (FIG. 2, step e). The deletion library is thenconverted into a fragment library (14 base-pair fragments 26 in thiscase) by digestion with restriction endonucleases BsmI and BpmI (FIG. 2,step f), purification of the plasmid containing the 14 bp fragment 26from the excised BpmI/BsmI fragment 30 (FIG. 2, step g), end-polishingwith T4 DNA polymerase (FIG. 2, step h), and ligation with T4 DNA ligase(FIG. 2, step i). Not stated, but implied, after each ligation step(i.e., steps a, e, and i) the ligation mixture is transformed intobacteria, the DNA is recovered from the bacteria, and the recovered DNAis used in the subsequent step. All of these reactions involvingrestriction endonucleases, ligases, polymerases, nucleases, and the likeare well known in the art and are performed according to standardmethods, e.g., J. Sambrook et al., Molecular Cloning: A LaboratoryManual (2d ed., 1989); T. Maniatis et al., Molecular Cloning: ALaboratory Manual (1982); F. Ausubel et al., Current Protocols inMolecular Biology (1987), relevant parts of which are herebyincorporated by reference.

The essence of the procedure is as follows. A gene of interest isconverted into a library of fragments serially deleted after everynucleotide. This deletion library is subsequently converted into afragment library containing all overlapping fragments encoded by thegene.

The fragment library can also serve as the starting point forconstruction of other types of antisense libraries. One such library isan antisense hammerhead ribozyme library.

A hammerhead ribozyme 34 is a small RNA that can catalyze the cleavageof a complementary RNA target 38 (FIG. 3). The hammerhead comprises acatalytic core 42 (SEQ ID NO:4), essential for cleavage activity.Additionally, the hammerhead has a recognition domain 46 that isrequired for interaction with a complementary substrate, such as an RNAtranscript. There are few sequence requirements for the recognitiondomain, thus by changing the sequence of the recognition domain almostany sequence can be targeted for cleavage by the hammerhead. Cleavage ofthe substrate 38 occurs at a cleavage site 50 containing an NUH sequence(where N is A, C, G, or U and H is A, C, or U). In the case where thesubstrate is a gene transcript, the hammerhead can be used as anantisense inhibitor of gene expression.

To convert the fragment (antisense RNA) library into a hammerheadribozyme library, a DNA fragment encoding the hammerhead catalytic coreis inserted into the DNA fragment encoding the antisense RNA. This isperformed as illustrated in FIG. 4. The 14 base-pair fragment 54 in thefragment library is bisected with HphI (FIG. 4, step a). The resultingsingle-stranded overhang on each terminus is then removed using the 3′to 5′ exonuclease activity of T4 DNA polymerase (FIG. 4, step b) toresult in blunt ends 58, 62. A DNA fragment encoding the hammerheadcatalytic core 66 (SEQ ID NO:4) is then inserted by ligation (FIG. 4,step c). The catalytic core shown in FIG. 4 is interrupted by apromoter-less chloramphenicol resistance gene 70 (CAT). A promoter isprovided flanking the MCS. Transforming bacteria and selecting forchloramphenicol resistance allows selection for clones in which thecatalytic core is in the correct orientation to produce a bonafidehammerhead ribozyme. Next, the CAT gene is removed and the sequenceencoding a hammerhead ribozyme 74 is generated by NruI digestion (FIG.4, step d) and ligation with T4 DNA ligase (FIG. 4, step e).

Other types of antisense libraries can also be produced from thefragment library. For instance, other cassettes can be ligated into anHphI-digested fragment library. Catalytic cores from other ribozymes,including those currently known and those to be discovered, can beinserted. Additionally, other cassettes could be used that encodesequences that cause modification to the target by mechanisms other thancleavage. Similarly, ribozyme and non-ribozyme sequences can be added tothe end of the antisense sequence. This is illustrated in FIG. 5A,wherein the DNA fragment library is digested with BpmI, which digeststhe DNA at the distal end of the inserted fragment 78 (step a). Theunpaired nucleotides resulting from this reaction are then removed withT4 DNA polymerase (step b) to result in blunt ends 82, 86. Next, acassette 90 is inserted by ligation to recircularize the modifiedplasmid 94, now containing the cassette inserted at an end of the insertfragment. Alternatively, instead of inserting a cassette after thefragment library is produced, a suitable cassette can be engineered intothe starting multi-cloning sequence. For instance, the HphI site of theoriginal MCS (FIG. 1A) could be replaced with a cassette encoding anydesired sequence (FIG. 5B). Then, using the same procedure illustratedin FIG. 2, the cassette can be placed against the fragment sequence inthe conversion of the deletion library into the fragment library. Anexample of a possible cassette is one encoding the sequence CUGA. Anantisense RNA with this sequence at its 3′ end has been shown to becapable of directing the 2′-O-methylation of the complementary target(J. Cavaille et al., Targeted ribose methylation of RNA in vivo directedby tailored antisense RNA guides, 383 Nature 732-735 (1996)). Thisreaction is catalyzed by modification machinery present in mammaliancells. 2′-O-methylation of a suitable target site could be used toinhibit expression of the RNA transcript. Other cellular RNA processingreactions can also be used in a similar fashion with the use ofdifferent cassettes placed adjacent to the antisense RNA sequence.

Use of Directed Libraries in the Identification of Target Sites forAntisense-Mediated Gene Inhibition

Antisense libraries prepared according to the present invention can beassayed in vitro in a cell free system or in vivo in cultured cells, aswill be described in more detail below.

In vivo assay. For in vivo, use the antisense library is introduced bytransfection into a suitable cell line that expresses the gene ofinterest. The transfection conditions are chosen such that only onemember of the library is taken up by each individual cell. Theindividual cells then each express a different antisense moleculetargeted to a different site on the RNA transcript of interest. Alltarget sites are represented in the entire cell population produced bytransfection. Using a suitable detection method, cell clones can beidentified in which expression of the target RNA has been reduced oreliminated. These clones possess an antisense molecule that targets aneffective site on the RNA transcript of interest. The plasmid encodingthis antisense molecule is recovered and the target sequence isidentified by DNA sequencing.

To identify suitable targets in vivo, specially designed expressionvectors are required. One key feature of such expression vectors is thatthey are designed to replicate episomally in mammalian cells. FIGS. 6Aand 7B show two such episomal vectors, pBK (SEQ ID NO:17) and pShuttle(SEQ ID NO:14), respectively. Vector pBK possesses the origin ofreplication and the gene encoding the T/t antigen from the human papovavirus BK (BKV). Vector pShuttle possesses the origin of replication andthe EBNA1 gene from the human Epstein-Barr virus (EBV). These sequenceelements allow each of the plasmids to replicate extrachromosomally(episomally). Episomal expression is desirable for several reasons.First, it eliminates the clone-to-clone variation in expression thatoccurs if stable transfectants are used P. B. Belt et al., 84 Gene407-417 (1989). Second, since the copy number of the episomal vector isdetermined primarily by the transfection conditions and, onceestablished, remains tightly regulated, J. L. Yates & N. Guan, 65 J.Virol. 483-488 (1991), then effects on expression due to differences incopy number are minimal. Consequently, the selection of antisenseefficacy is based on accessibility and not the level of expression.Third, the use of an episomal expression vector allows for hightransfection efficiency. P. B. Belt et al., 84 Gene 407-417 (1989); R.F. Maragolskee et al., 8 Mol. Cell. Biol. 2837-2847 (1988). This isimportant to ensure that all antisense agents present in the library arerepresented in the mammalian transfectants. Finally, the plasmid can berecovered and shuttled back into bacterial cells. This allows thesequence of effective antisense agents to be determined, therebyidentifying accessible target sites. As a demonstration of episomalreplication, pShuttle was used to transfect HeLa cells, and the cellswere grown in culture under 400 μg/ml hygromycin selection. After 1month in culture, low molecular weight DNA was isolated from 1×10⁷ cellsand used to transform Escherichia coli DH5α, producing a total of 2475hygromycin-resistant colonies.

Vector pBK illustrates other features of value for in vivo expression ofantisense libraries. pBK has a single antibiotic resistance gene,bleomycin^(R), driven by dual mammalian (CMV) and bacterial (em7)promoters. This allows the same selectable marker to be used in bothbacterial and mammalian cells. This helps to minimize the size of thevector, since large vectors transfect at a lower efficiency. pBK hasboth the BK origin of replication and the origin of replication from thepUC series of bacterial plasmids. Therefore pBK can be replicated inboth bacterial and mammalian cells, and can be shuttled between them.pBK was designed such that the antisense library could be constructedand expressed from the same vector. The antisense sequence is expressedby read-through expression of the bleomycin^(R) gene. This ensuresexpression of the antisense agent when the cells are grown in thepresence of bleomycin. The antisense fragment is released from thelarger bleomycin transcript by the activity of cis-acting ribozymes(CAR), hammerhead ribozymes in this case, that flank the antisensesequence. In the absence of CAR, flanking sequences of the largerbleomycin transcript could inhibit the activity of the antisense agent.Sequences outside of the MCS (FIG. 1A) encode the cis-acting ribozymes.They are illustrated in FIG. 6B where only the sequence of the upperstrand of the MCS is shown (SEQ ID NO:18). On cleavage by the CAR, theantisense agent is released and stable hairpin loops form to increasethe nuclease resistance of the antisense agent.

pShuttle shares many of the same features as pBK, with two significantdifferences. First, this episomal vector is EBV-based rather thanBKV-based. The second and more significant difference is thatconstruction of the antisense library is not possible in pShuttle.Instead, the antisense library is first constructed in pASlib (SEQ IDNO:7), and subsequently transferred to pShuttle for expression inmammalian cells. The antisense encoding fragment of pASlib is removed bydigestion with HindIII and SalI (FIG. 7A). Subsequently, theHindIII/SalI fragment is ligated into the multi-cloning site of pShuttlevia the HindIII and XhoI sites (FIG. 7C). This places the antisensesequence downstream of a dual CMV/T7 promoter for expression in vivo inmammalian cells or, alternatively, in vitro by transcription using T7RNA polymerase.

Although it is believed that episomal shuttle vectors are advantageousfor expression of directed antisense libraries, viral vectors can alsobe used. Many viruses are currently being examined for expression offoreign genes for the purpose of gene therapy. These same viral vectorswould be suitable for expression of directed antisense libraries. Someof these vectors replicate extrachromosomally and therefore behavesimilarly to the described episomal vectors. Others integrate intochromosomes. For the use of integrative viral vectors, two minorproblems would need to be dealt with. First, the antisense gene presentwithin the viral vector would integrate into the chromosome with thevirus. Consequently, recovering the gene to determine the site at whichit targets is not readily possible. This can be dealt with by usingpolymerase chain reaction (PCR) to amplify the integrated antisensegene. The PCR product could be sequenced directly, or cloned andsequenced to identify the target site. Second, some of these viralvectors integrate randomly and this would produce differing levels ofexpression from different members of the directed antisense library. Asdiscussed, it is important that expression of all members of the librarybe comparable. This problem can be dealt with by using a viral vectorthat integrates at a specific preferred site, such as adeno-associatedvirus.

In vitro assay. Identification of effective antisense target sites usingantisense libraries can also be performed using in vitro assays. Forinstance, an assay such as that used by Lieber and Strauss (A. Lieber &M. Strauss, Selection of efficient cleavage sites in target RNAs byusing a ribozyme expression library, 15 Molecular and Cellular Biology540-551 (1995)), can be used. For this, the antisense library isproduced by in vitro transcription from a suitable promoter. In thepresent case, an antisense ribozyme library in pShuttle might be used.Of course other types of antisense libraries could be used similarly.The library-containing pShuttle is digested with XbaI and used as atemplate for run-off transcription of the antisense ribozyme by in vitrotranscription with T7 RNA polymerase, according to methods well known inthe art (e.g., C. J. Noren et al., 18 Nucleic Acids Res. 83-88 (1990).Subsequently, the transcribed ribozyme library is incubated in a lysateprepared from a mammalian cell line expressing the gene of interest.Effective target sites are identified by performing a primer extensionreaction on purified RNA from the lysate using a primer specific for thegene of interest. Primer extension products terminate at the sites ofcleavage by effective ribozymes. These sites are identified by gelelectrophoresis of the primer extension products with suitable sizemarkers.

EXAMPLE 1

Construction of pASlib. In this example, there is described anillustrative plasmid according to the present invention for making adeletion library of a selected DNA. This plasmid was constructed asfollows.

The HindIII-HpaI fragment of pLA2917 (J. N. Allen & R. S. Hanson, 161 J.Bact. 955-962 (1985)), containing the kanamycin resistance gene, wasinserted into HindIII/SmaI-digested pUC19 to produce pUCKan. An HphI andtwo BsaHI sites were eliminated from the kanamycin resistance gene bysite-directed mutagenesis, according to methods well known in the art,to produce pUCKan*. The mutagenized kanamycin resistance gene wasremoved by HindIII/EcoRI digestion, and the termini were blunted by5′-overhang fill-in using the Klenow fragment of DNA polymerase I andligated to the 843 bp BspHI-SapI fragment of pUC19 containing the originof replication. A clone (pKan) was selected wherein the EcoRI and BspHIsites were juxtaposed. The BsmFI and PstI sites were eliminated frompKan by site-directed mutagenesis using the procedure of E. Merino etal., 12 Biotechniques 508-510 (1992). The multiple cloning site forpASlib was constructed from the overlapping oligodeoxynucleotides MCS-L(SEQ ID NO:5) and MCS-R (SEQ ID NO:6) by 5′-overhang fill-in with theKlenow fragment of DNA polymerase I. Oligonucleotides were synthesizedusing an Applied Biosystems automated oligonucleotide synthesizer. Thedouble-stranded multiple cloning site was inserted into EcoRI-linearizedand blunted pKan to result in pASlib (SEQ ID NO:7). Restrictionendonuclease digestions, primer extension reactions, ligation reactions,and the like were carried out according to methods well known in theart. E.g., J. Sambrook et al., Molecular Cloning: A Laboratory Manual(2d ed., 1989); T. Maniatis et al., Molecular Cloning: A LaboratoryManual (1982); F. Ausubel et al., Current Protocols in Molecular Biology(1987).

Therefore, pASlib possesses the pUC19 origin of replication and akanamycin resistance gene allowing selection in bacterial cells. Thekanamycin resistance gene was chosen as the selectable marker since, ofall the available bacterial selection markers, it possessed the fewestsites present in the MCS. Therefore, it was the simplest to modify bysite-specific mutagenesis to eliminate the undesirable sites. The MCScontains the following salient features. It possesses a short polylinkerthat allows much flexibility in the cloning of the gene or cDNA sequenceof interest, which represents the first step in the construction of anantisense library. The polylinker includes several restriction sitesthat leave sticky ends upon digestion. These sites can be used todirectionally clone the cDNA or genomic fragment in the correctorientation. Alternatively, the fragment can be cloned by blunt-endligation, and the correctly oriented clone can be selected byrestriction analysis. The PstI and PmeI sites allow the generation of asubstrate for unidirectional digestion by exonuclease III into thecloned cDNA or genomic fragment. This allows preparation of a serialdeletion library of the cloned insert. The BsmFI and BbsI sites are usedtogether to convert the deletion library into a 14 bp fragment library.The HphI site allows bisection of the 14 bp fragment library forintroduction of the antisense agent.

EXAMPLE 2

Construction of pShuttle. In this example, there is described theconstruction of an illustrative plasmid for use according to the presentinvention for expressing an antisense agent in either mammalian cellsusing the intermediate-early promoter from cytomegalovirus or in vitrousing T7 polymerase.

A hygromycin expression cassette capable of being expressed in bothmammalian and prokaryotic systems was constructed using overlapextension PCR. PCR was carried out according to methods well known inthe art, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;4,965,188; PCR Technology: Principles and Applications for DNAAmplification (H. Erlich ed., Stockton Press, New York, 1989); PCRProtocols: A guide to Methods and Applications (Innis et al. eds,Academic Press, San Diego, Calif., 1990). The 1026 bp hygromycinresistance coding sequence from EBOpLPP (ATCC) was joined at its 3′-endto the 322 bp 3′-untranslated region (UTR)/SV40 early polyadenylationsequence from pRC/CMV (Invitrogen Corp., Carlsbad, Calif.), while the527 bp dual ampicillin/SV40 early promoter from pEGFP-1 was joined tothe 5′-end. The sequences of the primers used in the PCR were asfollows: 3′-UTR/poly(A) segment, SEQ ID NO:8 and SEQ ID NO:9; hygromycincoding region, SEQ ID NO:10 and SEQ ID NO:11; amp/SV40 early promoter,SEQ ID NO:12 and SEQ ID NO:13. Each portion of the hygromycin cassettewas prepared by PCR using one of the three primer sets and theappropriate template. The resulting fragments were gel purified. Thehygromycin-encoding and the 3′-UTR/poly(A) fragments were combined andused in a second PCR reaction to produce the hygromycin-3′-UTR/poly(A)fragment. In a final PCR, this fragment was combined with the amp/SV40fragment to produce the complete 1875 bp hygromycin gene cassette. Thehygromycin gene cassette was ligated into the 843 bp BspHI-SapIoriP-containing fragment of pUC19, producing pHyg. The 4914 bpEcoRI-BamHI fragment containing the EBNA-1 and EBV oriP sequences fromEBOpLPP was inserted between the hygromycin cassette and the pUC19origin of XhoI-digested pHyg to make pEBV. The 1060 bp expressioncassette was excised from pRC/CMV using NruI and PvuII and inserted intothe BamHI site of pEBV to produce pShuttle (SEQ ID NO: 14).

pShuttle was designed to allow replication and expression of theantisense library in mammalian cells. It possesses an MCS for insertionof the antisense library. The MCS is flanked on one end by a dual CMV/T7promoter for allowing expression of the antisense agent gene both inmammalian cells as well as by in vitro transcription using T7 RNApolymerase. On the other end of the MCS is a bovine growth hormonepolyadenylation signal for efficient expression in mammalian cells.pShuttle possesses a hygromycin resistance gene driven by a dualpromoter for allowing selection in bacterial and mammalian cells. ThepUC19 origin of replication allows replication in bacterial cells. Forreplication in mammalian cells, the EBV origin and EBNA-1 gene wereincluded. J. Yates et al., 81 Proc. Nat'l Acad. Sci. USA 3806-3810(1984); J. Yates et al., 313 Nature 812-815 (1985).

EXAMPLE 3

Construction of a hammerhead ribozyme catalytic core cassette. Acassette encoding the hammerhead catalytic core, interrupted by the CATgene (S. Horinouchi & B. Weisblum, 150 J. Bact. 815-825 (1982)), wasconstructed as follows. PCR primers were prepared that werecomplementary to the CAT gene on their 3′-ends and encoded thehammerhead catalytic core on their 5′-ends. The sequences of the primerswere SEQ ID NO:15 and SEQ ID NO:16. Located between the CAT andhammerhead catalytic core sequences were NruI restriction sites. The PCRcontained 5 ng CAT gene DNA, 100 pmol each of the primers CatCass 1 andCatCass 2, 1 mM of each of the four dNTPs, 5 units of VENT polymerase(New England Biolabs, Beverly, Mass.) in the standard VENT polymerasebuffer except that the concentration of the MgSO₄ was increased to 5.2mM (i.e., 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.8 at 24° C.),5.2 mM MgSO₄, 0.1% Triton X-100). The use of VENT polymerase ensuredthat the cassette possessed blunt ends.

The reaction mixture was incubated as follows:

(A) 2 minutes at 94° C.;

(B) 5 cycles of 1 minute at 94° C., 30 seconds at 45° C., and 2 minutesat 72° C.;

(C) 15 cycles of 30 seconds at 94° C., 15 seconds at 60° C., and 2minutes at 72° C.; and

(D) 5 minutes at 73° C.

After amplification, the cassette was purified from unincorporatedprimers by agarose gel electrophoresis, and the agarose was subsequentlyremoved from the cassette DNA using standard procedures.

Introduction of a catalytic core into a fragment library presentsseveral difficulties. The core must be inserted by blunt-end ligationand in the correct orientation to produce a functional ribozyme.Additionally, due to its small size, it is difficult to prevent theintroduction of concatamers of the core and/or contamination of thelibrary with clones that do not acquire a catalytic core. To increasethe effectiveness and efficiency of this step, the core interrupted bythe CAT gene was designed. CAT selection allows the use of anon-phosphorylated cassette. This prevents insertion of multimers andselects against non-recombinants. Additionally, the CAT gene allowsselection of clones acquiring a correctly oriented catalytic core. Inthe desired orientation, transcription of the CAT and kanamycin genes isin the same direction. In the incorrect orientation, CAT expression isinhibited by antisense expression from the kanamycin resistance gene.This phenomenon has been noted previously, R. Bruckner et al., 32 Gene151-1160 (1984). After selection, the CAT gene is removed by digestionwith NruI to produce a sequence encoding a hammerhead ribozyme.

EXAMPLE 4

Construction of a Herpes ICP4 ribozyme library. The 4489 bp BglII-EcoRIfragment from pTEG2, X.X. Zhu et al., 184 Virology 67-78 (1991),containing a herpes simplex virus ICP4 genomic fragment was cloned intoEcoRI-EcoRV-digested pASlib. This fragment included 125 bp upstream ofthe translational start site, 466 bp downstream of the translationaltermination sequence, and the entire genomic coding sequence of ICP4.The resulting clone, pASlib-ICP4, contained the ICP4 fragment with thesense strand as the upper strand.

From pASlib-ICP4 a deletion library was produced as follows. Twenty μgof CsCl gradient purified plasmid DNA was digested with PstI and XbaI,then concentrated and desalted using a Microcon 50 spin filter (Amicon).The DNA was brought up to a volume of 60.4 μl of exonuclease III buffer(i.e., 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 10 mM 2-mercaptoethanol),warmed to 37° C., and then 300 units of exonuclease III were added. At1-minute intervals after the addition of the exonuclease III, 2.5 μl ofthe reaction mixture was removed and placed in microfuge tubes on icecontaining 7.5 μl of 66.7 mM sodium acetate, pH 5.2, 200 mM NaCl, 1.3 mMZnCl₂, and 1 unit of mung bean nuclease. After 25 aliquots had beenremoved, the mung bean nuclease-containing tubes were incubated at 20°C. for 30 minutes. After 30 minutes, the contents of all of the mungbean nuclease-containing tubes were combined and extracted withphenol-chloroform (50:50), extracted with chloroform, and then wereprecipitated with two volumes of 100% ethanol. The DNA was then pelletedand dried. The DNA was then resuspended in 18 μl of d.i. H₂O, 2.5 μl of10 mM of each of the four dNTPs, 2.5 μl of 10×Pfu polymerase buffer(e.g., 100 mM KCl, 100 mM (NH₄)₂SO₄, 200 mM Tris-Cl (pH 8.75), 20 mMMgSO₄, 1% Triton® X-100, 1000 mg/ml BSA), and 5 units of Pfu polymerase(Stratagene, La. Jolla, Calif.), then incubated at 72° C. for 15minutes, and cooled to room temperature. The plasmid DNA wasrecircularized by ligating in a large volume (1.25 ml) in a buffercontaining 5% PEG for 4 hours at room temperature. Except for themodifications indicated, all ligations were performed with T4 DNA ligaseunder the conditions suggested by the manufacturer.

After transformation into E. coli DH5α, the cells were grown in liquidculture to amplify the deletion library. The library DNA was purifiedand digested with BsmFI and BbsI, and then the ends were blunted withPfu polymerase as described above. The DNA was then recircularized byligation in a 600 μl volume in a buffer containing 5% polyethyleneglycol (PEG) at room temperature for 4 hours. After transformation intoE. coli DH5α, amplification and plasmid purification, 1 μg of libraryplasmid DNA was then subjected to digestion with 8 units of HphI for 1hour at 37° C., and the ends were polished with T4 DNA polymerase. Thehammerhead core cassette was inserted by ligating 0.5 μg of theHphI-digested library DNA with 5 μg of the ribozyme core sequencecassette prepared according to the procedure of Example 3. That ligationproduct was transformed into DH5α and grown in culture underchloramphenicol selection. After purification, 2 μg of the DNA wasdigested with HindIII and SalI, and the terminal phosphates were removedusing shrimp alkaline phosphatase (Amersham, Arlington Heights, Ill.).The HindIII/SalI digest was fractionated on an agarose gel, and thedephosphorylated ribozyme/chloramphenicol cassette was purified usingstandard procedures. The cassette was combined with an equimolar amountof HindIII-XhoI-digested pShuttle, prepared according to the procedureof Example 2, and ligated using a modified two-step ligation procedure,S. Damak & D. W. Bullock, 15 Biotechniques 448-450 (1993) (herebyincorporated by reference). The first step was performed at roomtemperature for 1 hour, and the second step was incubated overnight at16° C. The ligation mixture was transformed in DH5α and grown in cultureunder chloramphenicol selection. The library DNA was purified and thendigested with NruI to release the chloramphenicol gene. The digested DNAwas then recircularized by ligation in a volume of 600 μl. The finalligation product was transformed into DH5α, and the plasmid DNA waspurified on a CsCl gradient.

To verify the effectiveness of this procedure, 56 clones obtained atvarious steps were sequenced. Thirty-one were from the final ribozymelibrary, and the remainder were from earlier steps, beginning with the14 bp fragment library. The results of the sequencing are discussedbelow.

One observation made after the mung bean digestion was that thedeletions infrequently stopped at A-T base pairs. While exonuclease IIIhas been shown to exhibit a preference for stopping at certainnucleotides (C>A=T>G), W. Linxweiler & W. Horz, 10 Nucleic Acids Res.4845-4859 (1982), this was not believed to be the cause of the observedsequence bias. Instead, it is believed that this is the result of agreater degree of “breathing” at A-T terminated deletions and thesubsequent removal of A-T terminal pairs by mung bean nuclease. The mungbean nuclease digestion was later performed at higher saltconcentrations (150 mM) and at a lower temperature (20° C.). Thiseliminated the under-representation of A-T terminated deletions.

For construction of the library, two type IIS restriction enzymes arerequired, BsmFI and HphI. Typical of type IIS restriction enzymes, BsmFIand HphI cleave downstream of their recognition sequences in asequence-independent manner. Cleavage by type IIS restriction enzymescan pose some problems since they can exhibit infidelity in how far fromtheir recognition site they cleave. Cleavage by BsmFI was largely at theexpected distance (10/14), but also at 11/15. The reported 9/13 activityfor this enzyme, V. E. Velculescu et al., 270 Science 484-487 (1995),was not seen in any of the clones sequenced. Infidelity of BsmFI doesnot present a problem for construction of ribozyme libraries. The resultof this infidelity is that the recognition domains of the ribozymes inthe library can vary from 13 to 15 nucleotides.

In contrast, HphI infidelity can be problematical. HphI digestion is acritical step in the construction of ribozyme libraries. This enzymeproduces a 1 nucleotide 3′-overhang that is later removed by polishingwith T4 DNA polymerase. It is essential to the proper functioning of theresulting ribozyme that this 1 nucleotide be removed, since it does nothave an antisense binding partner in the ribozyme (FIG. 1, X).

HphI cleaves at 8/7, but also at 9/8. D. Kleid et al., 73 Proc. Nat'lAcad. Sci. USA 293-297 (1976). This infidelity is demonstrated in thepresent library by the presence of ribozymes with flanking helices oflength 8 and 5, as would be expected if HphI cleaved at 9/8. This typeof infidelity, in itself, is not problematical. It simply alters therelative lengths of the two arms of the binding domain, leaving thetotal length of the binding arms unchanged. The problem that arises withHphI infidelity is that the enzyme can cleave twice at the same target,i.e., if it first cleaves at 9/8 it can rebind and cleave at 8/7. Theresult is that 2 bp are removed from the sequence upon polishing with T4DNA polymerase. Removal of 2 bp from the insertion site of the ribozymecore produces a non-functional ribozyme. In early attempts to produce alibrary, >40% of the clones were the product of double cutting. This isclose to the statistically predicted 50% that would occur if HphI has nopreference for either 8/7 or 9/8 cutting. To minimize the possibility ofdouble cutting, HphI digestion was performed under near “single hit”conditions. Under these conditions, double cleavage was only 13% of thefinal library. It should be possible to further reduce the percentage ofdouble hits by performing the cleavage under “sub-single hit”conditions. This should not present any problem so long as the amount ofplasmid digested is sufficient to allow full representation of theribozyme library. Undigested molecules cannot accept the catalytic coreand are removed in the later step by selection for chloramphenicolresistance. Other class IIS restriction enzymes, such as MboII, couldlikely substitute for HphI, but fidelity may not be any better.

The infidelity of HphI raises another issue. It is possible that somesequences favor digestion at 8/7 and others at 9/8. This could lead tothe absence of some ribozyme target sequences in the final library. Thisappears to be unlikely, however. First, as discussed, under conditionsthat give nearly 100% cleavage by HphI, >40% of the molecules are cuttwice. This is close to the 50% predicted if HphI exhibits no preferencefor 8/7versus 9/8 cutting. Second, two clones that both contain the same14 bp sequence of ICP4, Rz8 and Rz9 (Table 1), are the products of 8/7and 9/8 cleavage, respectively. This suggests that the interveningsequence between the binding site and the cleavage site does not affectwhere HphI cleaves.

HphI is also sensitive to overlapping dam methylation. This is also trueof MboII. Since 2 nucleotides of the four base consensus sequence fordarn methylation are provided by the variable sequence of the cDNAinsert, mathematically {fraction (1/16)} of the clones in the 14 bpfragment library (6.25%) will not be cleaved with HphI and will beeliminated from the final ribozyme library. This can be prevented bypassage of the 14 bp fragment library in a dam⁻ strain prior to HphIdigestion.

TABLE 1 Clone (Position) Target Sequence^(a) SEQ ID NO: Rz1 (1754)cgacgccgcccgcc 19 Rz2 (1992) cugcgcgcguggcu 20 Rz3 (2045) gcgccugcgcgggg21 Rz4 (2252) cgccgccgacgcgc 22 Rz5 (2411) cccccuccccgcg 23 Rz6 (2517)guggcccugucgcg 24 Rz7 (2590) gccacacggcggcg 25 Rz8 (2729) cgccgcgcggugcg26 Rz9 (2729)^(b) cgccgcgcggugcg 27 Rz10 (2837) cccccugcgcgccuc 28 Rz11(2915) gguggugcuguacuc 29 Rz12 (3246) gggcccgcgguguc 30 Rz13 (3275)^(c)ccuggcgugcgagc 31 Rz14 (3569) ggggaccaccgacgccauggc 32 Rz15 (3680)cguggcgcuggggc 33 Rz16 (3842) cgggauucgcuggg^(d) 34 ^(a)The nucleotidein bold indicates the unbound nucleotide, i.e., position X in FIG. 3.^(b)Clone repeated 2 times. ^(c)Clone repcated 3 times. ^(d)Bona fideribozyme target.

The target locations of the 56 sequenced clones are illustrated in FIG.8. The histogram indicates that the target sites are fairly evenlydistributed across the entire ICP4 gene, with the exception that noclones were identified targeting the 5′- and 3′-termini. It is unlikelythat the library is devoid of members targeting these regions since thelibraries are prepared with complexities far exceeding the total numberof sites on the gene. It is even possible that target sites in theseregions are similarly represented as those identified by the sequencedclones. Due to the small number of clones sequenced, it is likely thatsome larger gaps in the data could be observed even for a uniformlyrepresented library, such as the gap between positions 966 and 1282.

Of the 56 sequences determined, 42 (75%) occurred only once, while fouroccurred multiple times (FIG. 8). Three were only mildlyover-represented, with two or three occurrences compared with the singleoccurrence for the majority of clones. The three positions were 2054 and3246, with two occurrences each, and position 2729, with threeoccurrences. One position, 3275, was significantly over-represented,occurring seven times. Five of the occurrences were observed within the32 clones sequenced from the final library, and the other two were foundat early stages of the construction. The over-representation ofparticular sites is likely caused by some local sequence and/orstructure in the DNA that either stalls exonuclease m or causes it tofall off the template. P. Abarzua & K. J. Marians, 81 Proc. Nat'l Acad.Sci. USA 2030-2034 (1984). Performing the exonuclease III deletion athigher temperatures might reduce this phenomenon if an inhibitorystructure is forming at certain sequences. Higher temperature alsoallows for more distributive activity from the enzyme, J. D. Hoheisel,209 Anal. Biochem. 238-246 (1993), which is desirable in this type ofexonuclease III digestion. While it is possible that the exonuclease IIIdigestion conditions may need to be optimized for each target cDNA,creating libraries larger than would be necessary to represent everyposition would ensure complete representation of all target sites.

Examination of the 31 clones obtained from the final library alloweddetermination of the overall effectiveness of the procedure. All 31possessed a catalytic core, demonstrating the effectiveness of the useof CAT selection for this purpose. Nineteen of the 31 clones *\(61%)contained sequences that could potentially be ribozymes if the sequencethat they targeted included the required NUH sequence at the correctlocation. These are shown in Table 1. Counted among these potentialribozymes were three clones that possess non-detrimental defects. Onehas a single nucleotide deleted from loop II of the ribozyme (Rz13).This produces a three-, instead of a four-nucleotide loop II. The siteof this defect is the NruI site used to remove CAT from the catalyticcore. The ends must have been damaged during this step for this clone.The other two non-detrimental defects were the result of incompletedigestion by BsmFI. These clones have a longer flanking armcorresponding to helix III (Rz12 and Rz14). This appears to be theresult of a lack of cleavage of the BsmFI site on pASlib and, instead,an internal BsmFI site on ICP4 was used. These clones would be expectedto produce functional ribozymes had they targeted an NUH sequence.

The remaining 12 clones (39% of 31) possessed defects that would preventthem from being potentially functional ribozymes. Four of these (13%)were defective in that they were cleaved twice with HphI. As discussedabove, it is likely that this defect can be reduced to close to zero byperforming the HphI digestion under “sub-single hit” conditions. Three(9.7%) were missing 1 nucleotide from one end of the catalytic core.Since the deletion always occurred at the same end of the cassette andthe thermostable polymerase used to make the cassette does not containany 5′ to 3′ exonuclease activity, the PCR primer constituting that endof the cassette must have been contaminated with a small percentage of afailure fragment of the DNA synthesis. This defect can be eliminated bybetter purification of the primers. Five clones (16%) possessed thecatalytic core in the incorrect orientation. This is in contrast to theexpected 50% if there were no selection for orientation. Incorrectlyoriented clones could be eliminated by moving the promoter for the CATgene outside the MCS of pASlib. Finally, three clones were the result ofvarious unknown cloning artifacts.

Therefore, the success rate of this library was 61%. As discussed, a fewprocedural changes would increase the success rate to 70-80%. This couldbe increased a further 16% by placing the CAT promoter outside the MCS.Even at 61%, the success rate is more than adequate. This just meansthat it is necessary to screen an antisense library 140% the size neededif 100% success were achieved. This would still be a small libraryrelative to a non-directed library approach.

Three of the 31 clones (9.7%) targeted a site on the ICP4 mRNA thatcontained a uridine at the proper position of the consensus NUH site(Rz3, Rz5, and Rz16). Of the three, only one targeted a consensus NUHsite (Rz16). Due to the unusually high G/C content of the ICP4 genomicfragment used to make the ribozyme library, only 9.2% of the nucleotidesin the mRNA are uridines, of which 203 occur as an NUH triplet. The factthat the percentage of sequenced clones in the library targeting an NUsite is virtually identical to the percentage of uridines in the ICP4gene suggests that the library is unbiased and likely to contain afairly uniform distribution of target sites.

The use of a direct library for target site selection significantlysimplifies the screening process, since only very small libraries needbe prepared and assayed. For ICP4, assuming the library contains auniform distribution of the 4475 distinct sequences (4489-14), a libraryof 67,125 (15-fold excess) is expected to have a probability of 99.9% ofcontaining all sequences. W. Feller, An Introduction to ProbabilityTheory and Its Applications (3d ed. 1968). Based on a χ² goodness-of-fitanalysis of the 56 sequences, the multiples observed at positions 2729and 3275 occur with a higher frequency than would be expected for auniform distribution. All other positions are consistent with a uniformdistribution. Correcting for the two over-represented sequences, alibrary of 81,057 (18-fold excess) is expected to contain all sequenceswith probability of 99.9%. Preparation, manipulation, and screening ofsuch a library is well within the limitations of current practice. Incontrast, a non-directed library targeting 14 nucleotides would requirea minimum size of 2.7×10⁸ (4¹⁴). The ability to prepare and screen sucha library is questionable. Even if possible, the vast majority ofmembers of the library are directed at non-target genes. Inhibition ofnon-target genes could pose problems in interpreting the results.

50 1 62 DNA Artificial Sequence Multiple cloning site for use in makingdeletion libraries. 1 gcttggtgat gcattcgata tcgtttaaac gcccgggcgcggccgcggcg 50 cctccagtcg ac 62 2 24 DNA Artificial Sequence Portion of amultiple cloning site for use in making deletion libraries. 2 gtcgacgggactgcaggttt aaac 24 3 23 DNA Artificial Sequence Portion of a multiplecloning site for use in making deletion libraries. 3 gaagacagtcaccaagcttc agc 23 4 23 DNA Unknown Catalytic core of hammerheadribozyme. 4 ctgatgaggt cgcgagaccg aaa 23 5 49 DNA Artificial SequencePCR primer for construction of pASlib. 5 aagcttggtg actgtcttcgagctcgaatt catcgatatc tagagttta 49 6 34 DNA Artificial Sequence PCRprimer for construction of pASlib. 6 gtcgacggga ctgcaggttt aaactctagatatc 34 7 2077 DNA Artificial Sequence pASlib 7 tcagtggaac gaaaactcacgttaagggat tttggtcatg aattgtcgac 50 gggactgcag gtttaaactc tagatatcgatgaattcgag ctcgaagaca 100 gtcaccaagc ttattcccag agtcacgctc agaagaactcgtcaagaagg 150 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac200 gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg 250tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag 300 tcgatgaatccagaaaagcg gccattttcc accatgatat tcggcaagca 350 ggcatcgcca tgggtcacgacgagatcctc gccgtcgggc atgcgcgcct 400 tgagcctggc gaacagttcg gctggcgcgagcccctgatg ctcttcgtcc 450 agatcatcct gatcgacaag accggcttcc atccgagtacgtgctcgctc 500 gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg550 tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga 600gcaagatgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 650 ccaatcccttcccgcttcag tgacaacgtc gagcacagct gcgcaaggaa 700 cgcccgtcgt ggcaagccacgatagccgcg ctgcctcgtc ttgcagttca 750 ttcagggcac cggacaggtc ggtcttgacaaaaagaaccg gccgcccctg 800 cgctgacagc cggaacacgg cggcatcaga ggagccgattgtctgttgtg 850 cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg900 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 950atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca 1000 tccagtttactttgcagggc ttcccaacct taccagaggt cgccccagct 1050 ggcaattccg gttcgcttgctgtccataaa accgcccagt ctagctatcg 1100 ccatgtaagc ccactgcaag ctacctgctttctctttgcg cttgcgtttt 1150 cccttgtcca gatagcccag tagtgacatt catccggggtcagcaccgtt 1200 tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc1250 cctgagtgct tgcggcagcg tgaagctgct tcctcgctca ctgactcgct 1300gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 1350 taatacggttatccacagaa tcaggggata acgcaggaaa gaacatgtga 1400 gcaaaaggcc agcaaaaggccaggaaccgt aaaaaggccg cgttgctggc 1450 gtttttccat aggctccgcc cccctgacgagcatcacaaa aatcgacgct 1500 caagtcagag gtggcgaaac ccgacaggac tataaagataccaggcgttt 1550 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac1600 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat 1650gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 1700 ggctgtgtgcacgaaccccc cgttcagccc gaccgctgcg ccttatccgg 1750 taactatcgt cttgagtccaacccggtaag acacgactta tcgccactgg 1800 cagcagccac tggtaacagg attagcagagcgaggtatgt aggcggtgct 1850 acagagttct tgaagtggtg gcctaactac ggctacactagaaggacagt 1900 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg1950 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 2000gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 2050 tttgatcttttctacggggt ctgacgc 2077 8 33 DNA Artificial Sequence PCR primer foramplifying a 3′-UTR/poly(A) segment. 8 ccgagggcaa aggaataggc gggactctggggt 33 9 21 DNA Artificial Sequence PCR primer for amplifying a3′-UTR/poly(A) segment. 9 ctcgaggtcg acgggatcca g 21 10 33 DNAArtificial Sequence PCR primer for amplifying a hygromycin codingregion. 10 ggatgaggat cgtttcgcat gaaaaagcct gaa 33 11 33 DNA ArtificialSequence PCR primer for amplifying a hygromycin coding region. 11accccagagt cccgcctatt cctttgccct cgg 33 12 20 DNA Artificial SequencePCR primer for amplifying an amp/SV40 early promoter. 12 cgtcaggtggcacttttcgg 20 13 33 DNA Artificial Sequence PCR primer for amplifyingthe amp/SV40 early promoter. 13 ttcaggcttt ttcatgcgaa acgatcctca tcc 3314 8705 DNA Artificial Sequence pShuttle 14 tcgagcatga ccaaaatcccttaacgtgag ttttcgttcc actgagcgtc 50 agaccccgta gaaaagatca aaggatcttcttgagatcct ttttttctgc 100 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctaccagcggtggtt 150 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt200 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 250gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 300 atcctgttaccagtggctgc tgccagtggc gataagtcgt gtcttaccgg 350 gttggactca agacgatagttaccggataa ggcgcagcgg tcgggctgaa 400 cggggggttc gtgcacacag cccagcttggagcgaacgac ctacaccgaa 450 ctgagatacc tacagcgtga gcattgagaa agcgccacgcttcccgaagg 500 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc550 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 600gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 650 ggggcggagcctatggaaaa acgccagcaa cgcggccttt ttacggttcc 700 tggccttttg ctggccttttgctcacatgt tctttcctgc gttatcccct 750 gattctgtgg ataaccgtat taccgcctttgagtgagctg ataccgctcg 800 ccgcagccga acgaccgagc gcagcgagtc agtgagcgaggaagccgtca 850 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt900 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 950tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc 1000 tgtggaatgtgtgtcagtta gggtgtggaa agtccccagg ctccccagca 1050 ggcagaagta tgcaaagcatgcatctcaat tagtcagcaa ccaggtgtgg 1100 aaagtcccca ggctccccag caggcagaagtatgcaaagc atgcatctca 1150 attagtcagc aaccatagtc ccgcccctaa ctccgcccatcccgccccta 1200 actccgccca gttccgccca ttctccgccc catggctgac taattttttt1250 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt 1300agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga 1350 gacaggatgaggatcgtttc gcatgaaaaa gcctgaactc accgcgacgt 1400 ctgtcgagaa gtttctgatcgaaaagttcg acagcgtctc cgacctgatg 1450 cagctctcgg agggcgaaga atctcgtgctttcagcttcg atgtaggagg 1500 gcgtggatat gtcctgcggg taaatagctg cgccgatggtttctacaaag 1550 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa1600 gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg 1650ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg 1700 ctgttctgcagccggtcgcg gaggccatgg atgcgatcgc tgcggccgat 1750 cttagccaga cgagcgggttcggcccattc ggaccgcaag gaatcggtca 1800 atacactaca tggcgtgatt tcatatgcgcgattgctgat ccccatgtgt 1850 atcactggca aactgtgatg gacgacaccg tcagtgcgtccgtcgcgcag 1900 gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca1950 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc 2000gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 2050 tacgaggtcgccaacatctt cttctggagg ccgtggttgg cttgtatgga 2100 gcagcagacg cgctacttcgagcggaggca tccggagctt gcaggatcgc 2150 cgcggctccg ggcgtatatg ctccgcattggtcttgacca actctatcag 2200 agcttggttg acggcaattt cgatgatgca gcttgggcgcagggtcgatg 2250 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg2300 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 2350gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaataggc 2400 gggactctggggttcgaaat gaccgaccaa gcgacgccca acctgccatc 2450 acgagatttc gattccaccgccgccttcta tgaaaggttg ggcttcggaa 2500 tcgttttccg ggacgccggc tggatgatcctccagcgcgg ggatctcatg 2550 ctggagttct tcgcccaccc caacttgttt attgcagcttataatggtta 2600 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac2650 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 2700tggatccgat gtacgggcca gatatacgcg ttgacattga ttattgacta 2750 gttattaatagtaatcaatt acggggtcat tagttcatag cccatatatg 2800 gagttccgcg ttacataacttacggtaaat ggcccgcctg gctgaccgcc 2850 caacgacccc cgcccattga cgtcaataatgacgtatgtt cccatagtaa 2900 cgccaatagg gactttccat tgacgtcaat gggtggactatttacggtaa 2950 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc3000 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 3050tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 3100 gctattaccatggtgatgcg gttttggcag tacatcaatg ggcgtggata 3150 gcggtttgac tcacggggatttccaagtct ccaccccatt gacgtcaatg 3200 ggagtttgtt ttggcaccaa aatcaacgggactttccaaa atgtcgtaac 3250 aactccgccc cattgacgca aatgggcggt aggcgtgtacggtgggaggt 3300 ctatataagc agagctctct ggctaactag agaacccact gcttaactgg3350 cttatcgaaa ttaatacgac tcactatagg gagacccaag cttggtaccg 3400agctcggatc cactagtaac ggccgccagt gtgctggaat tctgcagata 3450 tccatcacactggcggccgc tcgagcatgc atctagaggg ccctattcta 3500 tagtgtcacc taaatgctagagctcgctga tcagcctcga ctgtgccttc 3550 tagttgccag ccatctgttg tttgcccctcccccgtgcct tccttgaccc 3600 tggaaggtgc cactcccact gtcctttcct aataaaatgaggaaattgca 3650 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca3700 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 3750cggtgggctc tatggcttct gaggcggaaa gaaccaggat cccccgccgc 3800 cggacgaactaaacctgact acggcatctc tgccccttct tcgctggtac 3850 gaggagcgct tttgttttgtattggtcacg gggcagtgca tgtaatccct 3900 tcagttggtt ggtacaactt gccaactgggccctgttcca catgtgacac 3950 ggggggggac caaacacaaa ggggttctct gactgtagttgacatcctta 4000 taaatggatg tgcacatttg ccaacactga gtggctttca tcctggagca4050 gactttgcag tctgtggact gcaacacaac attgccttta tgtgtaactc 4100ttggctgaag ctcttacacc aatgctgggg gacatgtacc tcccaggggc 4150 ccaggaagactacgggaggc tacaccaacg tcaatcagag gggcctgtgt 4200 agctaccgat aagcggaccctcaagagggc attagcaata gtgtttataa 4250 ggcccccttg ttaaccctaa acgggtagcatatgcttccc gggtagtagt 4300 atatactatc cagactaacc ctaattcaat agcatatgttacccaacggg 4350 aagcatatgc tatcgaatta gggttagtaa aagggtccta aggaacagcg4400 atatctccca ccccatgagc tgtcacggtt ttatttacat ggggtcagga 4450ttccacgagg gtagtgaacc attttagtca caagggcagt ggctgaagat 4500 caaggagcgggcagtgaact ctcctgaatc ttcgcctgct tcttcattct 4550 ccttcgttta gctaatagaataactgctga gttgtgaaca gtaaggtgta 4600 tgtgaggtgc tcgaaaacaa ggtttcaggtgacgccccca gaataaaatt 4650 tggacggggg gttcagtggt ggcattgtgc tatgacaccaatataaccct 4700 cacaaacccc ttgggcaata aatactagtg taggaatgaa acattctgaa4750 tatctttaac aatagaaatc catggggtgg ggacaagccg taaagactgg 4800atgtccatct cacacgaatt tatggctatg ggcaacacat aatcctagtg 4850 caatatgatactggggttat taagatgtgt cccaggcagg gaccaagaca 4900 ggtgaaccat gttgttacactctatttgta acaaggggaa agagagtgga 4950 cgccgacagc agcggactcc actggttgtctctaacaccc ccgaaaatta 5000 aacggggctc cacgccaatg gggcccataa acaaagacaagtggccactc 5050 ttttttttga aattgtggag tgggggcacg cgtcagcccc cacacgccgc5100 cctgcggttt tggactgtaa aataagggtg taataacttg gctgattgta 5150accccgctaa ccactgcggt caaaccactt gcccacaaaa ccactaatgg 5200 caccccggggaatacctgca taagtaggtg ggcgggccaa gataggggcg 5250 cgattgctgc gatctggaggacaaattaca cacacttgcg cctgagcgcc 5300 aagcacaggg ttgttggtcc tcatattcacgaggtcgctg agagcacggt 5350 gggctaatgt tgccatgggt agcatatact acccaaatatctggatagca 5400 tatgctatcc taatctatat ctgggtagca taggctatcc taatctatat5450 ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc 5500taatttatat ctgggtagca taggctatcc taatctatat ctgggtagca 5550 tatgctatcctaatctatat ctgggtagta tatgctatcc taatctgtat 5600 ccgggtagca tatgctatcctaatagagat tagggtagta tatgctatcc 5650 taatttatat ctgggtagca tatactacccaaatatctgg atagcatatg 5700 ctatcctaat ctatatctgg gtagcatatg ctatcctaatctatatctgg 5750 gtagcatagg ctatcctaat ctatatctgg gtagcatatg ctatcctaat5800 ctatatctgg gtagtatatg ctatcctaat ttatatctgg gtagcatagg 5850ctatcctaat ctatatctgg gtagcatatg ctatcctaat ctatatctgg 5900 gtagtatatgctatcctaat ctgtatccgg gtagcatatg ctatcctcat 5950 gcatatacag tcagcatatgatacccagta gtagagtggg agtgctatcc 6000 tttgcatatg ccgccacctc ccaagggggcgtgaattttc gctgcttgtc 6050 cttttcctgc tggttgctcc cattcttagg tgaatttaaggaggccaggc 6100 taaagccgtc gcatgtctga ttgctcacca ggtaaatgtc gctaatgttt6150 tccaacgcga gaaggtgttg agcgcggagc tgagtgacgt gacaacatgg 6200gtatgcccaa ttgccccatg ttgggaggac gaaaatggtg acaagacaga 6250 tggccagaaatacaccaaca gcacgcatga tgtctactgg ggatttattc 6300 tttagtgcgg gggaatacacggcttttaat acgattgagg gcgtctccta 6350 acaagttaca tcactcctgc ccttcctcaccctcatctcc atcacctcct 6400 tcatctccgt catctccgtc atcaccctcc gcggcagccccttccaccat 6450 aggtggaaac cagggaggca aatctactcc atcgtcaaag ctgcacacag6500 tcaccctgat attgcaggta ggagcgggct ttgtcataac aaggtcctta 6550atcgcatcct tcaaaacctc agcaaatata tgagtttgta aaaagaccat 6600 gaaataacagacaatggact cccttagcgg gccaggttgt gggccgggtc 6650 caggggccat tccaaaggggagacgactca atggtgtaag acgacattgt 6700 ggaatagcaa gggcagttcc tcgccttaggttgtaaaggg aggtcttact 6750 acctccatat acgaacacac cggcgaccca agttccttcgtcggtagtcc 6800 tttctacgtg actcctagcc aggagggccc ttaaaccttc tgcaatgttc6850 tcaaatttcg ggttggaacc tccttgacca cgatgctttc caaaccaccc 6900tccttttttg cgcctgcctc catcaccctg accccggggt ccagtgcttg 6950 ggccttctcctgggtcatct gcggggccct gctctatcgc tcccgggggc 7000 acgtcaggct caccatctgggccaccttct tggtggtatt caaaataatc 7050 ggcttcccct acagggtgga aaaatggccttctacctgga gggggcctgc 7100 gcggtggaga cccggatgat gatgactgac tactgggactcctgggcctc 7150 ttttctccac gtccacgacc tctccccctg gctctttcac gacttccccc7200 cctggctctt tcacgtcctc taccccggcg gcctccacta cctcctcgac 7250cccggcctcc actacctcct cgaccccggc ctccactgcc tcctcgaccc 7300 cggcctccacctcctgctcc tgcccctcct gctcctgccc ctcctcctgc 7350 tcctgcccct cctgcccctcctgctcctgc ccctcctgcc cctcctgctc 7400 ctgcccctcc tgcccctcct gctcctgcccctcctgcccc tcctcctgct 7450 cctgcccctc ctgcccctcc tcctgctcct gcccctcctgcccctcctgc 7500 tcctgcccct cctgcccctc ctgctcctgc ccctcctgcc cctcctgctc7550 ctgcccctcc tgctcctgcc cctcctgctc ctgcccctcc tgctcctgcc 7600cctcctgccc ctcctgcccc tcctcctgct cctgcccctc ctgctcctgc 7650 ccctcctgcccctcctgccc ctcctgctcc tgcccctcct cctgctcctg 7700 cccctcctgc ccctcctgcccctcctcctg ctcctgcccc tcctgcccct 7750 cctcctgctc ctgcccctcc tcctgctcctgcccctcctg cccctcctgc 7800 ccctcctcct gctcctgccc ctcctgcccc tcctcctgctcctgcccctc 7850 ctcctgctcc tgcccctcct gcccctcctg cccctcctcc tgctcctgcc7900 cctcctcctg ctcctgcccc tcctgcccct cctgcccctc ctgcccctcc 7950tcctgctcct gcccctcctc ctgctcctgc ccctcctgct cctgcccctc 8000 ccgctcctgctcctgctcct gttccaccgt gggtcccttt gcagccaatg 8050 caacttggac gtttttggggtctccggaca ccatctctat gtcttggccc 8100 tgatcctgag ccgcccgggg ctcctggtcttccgcctcct cgtcctcgtc 8150 ctcttccccg tcctcgtcca tggttatcac cccctcttctttgaggtcca 8200 ctgccgccgg agccttctgg tccagatgtg tctcccttct ctcctaggcc8250 atttccaggt cctgtacctg gcccctcgtc agacatgatt cacactaaaa 8300gagatcaata gacatcttta ttagacgacg ctcagtgaat acagggagtg 8350 cagactcctgccccctccaa cagccccccc accctcatcc ccttcatggt 8400 cgctgtcaga cagatccaggtctgaaaatt ccccatcctc cgaaccatcc 8450 tcgtcctcat caccaattac tcgcagcccggaaaactccc gctgaacatc 8500 ctcaagattt gcgtcctgag cctcaagcca ggcctcaaattcctcgtccc 8550 cctttttgct ggacggtagg gatggggatt ctcgggaccc ctcctcttcc8600 tcttcaaggt caccagacag agatgctact ggggcaacgg aagaaaagct 8650gggtgcggcc tgtgaggatc agcttatcga tgataagctg tcaaacatga 8700 gaatt 870515 29 DNA Artificial Sequence CatCass1 15 ctgatgaggt cgcgactagtgttgacaat 29 16 27 DNA Artificial Sequence CatCass2 16 ttcggtctcgcgagcaggtt agtgaca 27 17 5658 DNA Artificial Sequence pBK 17 ctagttctggcgcagaacca tggcctttgt ccagtttaac tggggacaag 50 gccaagattc ctaggctcgcaaaacatgtc tgtcatgcac tttccttcct 100 gaggtcatgg tttggctgca ttccatgggtaagcagctcc tccctgtgag 150 tcatgcactt tccttcctga ggtcatggtt tggctgcattcccctgtgag 200 tcatgcactt tccttcctga ggtcatggtt tggctgcatt ccatgggtaa250 gcagctcctc cctgtggcct ttttttttat aatatataag aggccgaggc 300cgcctctgcc tccacccttt ctctcaagta gtaagggtgt ggaggctttt 350 tctgaggcctagcaaaacta tttggggaaa tccctattct tttgcaattt 400 ttgcaaaaat ggataaagttcttaacaggg aagaatccat ggagctcatg 450 gaccttttag gccttgaaag agctgcctggggaaatcttc ccttaatgag 500 aaaagcttat ttaaggaagt gtaaggaatt tcatcctgacaaagggggcg 550 acgaggataa aatgaagaga atgaatactt tgtataaaaa aatggagcag600 gatgtaaagg tagctcatca gcctgatttt ggaacttgga gtagctcaga 650ggtttgtgct gattttcctc tttgcccaga taccctgtac tgcaaggaat 700 ggcctatttgttccaaaaag ccttctgtgc actgcccttg catgctatgt 750 cagcttagat taaggcatttaaatagaaaa tttttaagaa aagagccctt 800 ggtttggata gattgctact gcattgactgcttcacacag tggtttggct 850 tagacctaac tgaagaaact ctgcaatggt gggtccaaataattggagaa 900 actcccttca gagatctaaa gctttaaggt aactaactta tatttagata950 aataataaaa tattaaaagg ccctaagtaa ttattttttt tataggtgcc 1000aacctatgga acagaagagt gggagtcctg gtggagttcc tttaatgaaa 1050 aatgggatgaagatttattt tgccatgaag atatgtttgc cagtgatgaa 1100 gaagcaacag cagattctcaacactcaaca ccacccaaaa aaaaaagaaa 1150 ggtagaagac cctaaagact ttccctctgatctacaccag tttcttagtc 1200 aagctgtatt tagtaataga acccttgcct gctttgctgtgtatactact 1250 aaagaaaaag ctcaaattct gtataaaaaa cttatggaaa aatattctgt1300 aacttttatt agtagacaca tgtgtgctgg gcataatatt atattctttt 1350taactccaca tagacataga gtttctgcaa ttaataattt ctgtcaaaag 1400 ctgtgtacctttagtttttt aatttgtaag ggtgttaata aggaatactt 1450 actatatagt gccttaactagagatccata ccatactata gaagaaagca 1500 ttcaaggggg cttaaaggag catgattttagcccagaaga gcctgaagaa 1550 acaaagcagg tgtcttggaa attaattact gagtatgcagtagagacaaa 1600 gtgtgaggat gtgtttttat tattaggtat gtatttagaa tttcaataca1650 atgtagagga gtgtaaaaag tgtcagaaaa aagaccagcc ttatcacttt 1700aagtatcatg aaaagcactt tgcaaatgct attatttttg cagaaagtaa 1750 aaatcaaaaaagtatttgtc agcaagcagt agatacagtt ttagctaaaa 1800 aaagagtaga tacccttcatatgaccaggg aagaaatgct aacagaaaga 1850 ttcaatcata tattagataa aatggatttaatatttggag ctcatggaaa 1900 tgctgtacta gaacaatata tggcaggtgt tgcttggctgcactgtttgc 1950 tacctaaaat ggattctgta atatttgatt ttttgcactg tattgttttc2000 aatgtaccta aaagaagata ctggttattt aaaggtccca ttgatagtgg 2050aaaaacaaca ctagctgccg ggttattaga tttgtgtggt ggtaaagcct 2100 taaatgtaaacctacccatg gaaaggctaa cctttgagct aggtgtagct 2150 atagatcagt acatggttgtttttgaagat gtaaaaggga caggagctga 2200 atcaaaggat ttgccttcag gacatggaataaacaattta gacagtttga 2250 gagattattt agatggaagt gttaaggtaa atttagaaaagaaacattta 2300 aacaaaagaa cccaaatatt tccaccaggc ttggttacaa tgaatgagta2350 tcctgtccct aaaaccctgc aagctagatt tgtaagacaa atagatttta 2400ggcccaaaat atatttaaga aaatccttac aaaactcaga gttcttactt 2450 gaaaaaagaattttacaaag tggaatgacc ttgttgctac tgctaatttg 2500 gtttaggcct gtagctgattttgcaactga tatacaatct agaattgttg 2550 aatggaagga aaggctggat tctgagataagtatgtatac tttttcaagg 2600 atgaaatata atatatgctt ggggaaatgt attcttgatattacaagaga 2650 agaggattca gaaactgaag actctggaca tggatcaagc actgaatccc2700 aatcacaatg ctcttcccaa gtctcagata cttcagcccc tgctgaagat 2750tcccaaaggt cagaccccca tagtcaagag ttgcatttgt gtaaaggctt 2800 tcagtgttttaaaaggccta aaacaccacc cccaaaataa cacaagctta 2850 aaagtggctt atacaaaagcagcatttatt aaatgtatat gtacaataaa 2900 agcacctgtt taaagcattt tggtttgcaattgtccctgt ttgtcaatat 2950 atcttatcat atctgggtcc cctggaagta actagatgatccgctgtgga 3000 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga3050 agtatgcaaa gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 3100ggctccccag caggcagaag tatgcaaagt aatagtaatc aattacgggg 3150 tcattagttcatagcccata tatggagttc cgcgttacat aacttacggt 3200 aaatggcccg cctggctgaccgcccaacga cccccgccca ttgacgtcaa 3250 taatgacgta tgttcccata gtaacgccaatagggacttt ccattgacgt 3300 caatgggtgg agtatttacg gtaaactgcc cacttggcagtacatcaagt 3350 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc3400 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 3450cagtacatct acgtattagt catcgctatt accatggcga tgcggttttg 3500 gcagtacatcaatgggcgtg gatagcggtt tgactcacgg ggatttccaa 3550 gtctccaccc cattgacgtcaatgggagtt tgttttggca ccaaaatcaa 3600 cgggactttc caaaatgtcg taacaactccgccccattga cgcaaatggg 3650 cggtaggcgt gtacggtggg aggtctatat aagcagagctggtttagtga 3700 accgtcagat ccgctagcgc taccggactc agatctcgag ctcaagctaa3750 tcatcggcat agtatatcgg catagtataa tacgactcac tataggaggg 3800ccaccatggc caagttgacc agtgccgttc cggtgcttac cgcgcgcgac 3850 gtcgccggagcggtcgagtt ctggaccgac cggctcgggt tctcccggga 3900 cttcgtggag gacgacttcgccggtgtggt ccgggacgac gtgaccctgt 3950 tcatcagcgc ggtccaggac caggtggtgccggacaacac cctggcctgg 4000 gtgtgggtgc gcggcctgga cgagctgtac gccgagtggtcggaggtcgt 4050 gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg4100 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc 4150gtgcacttcg tggccgagga gcaggactga ccgacgccga ccaacaccgc 4200 cggggggaggctaactgaaa cacggaagga gacaataccg gaaggaaccc 4250 gcgctatgac ggcaataaaaagacagaata aaacgcacgg tgttgggtcg 4300 tttgttcata aacgcggggt tcggtcccagggctggcact ctgtcgatac 4350 cccaccgacg gcggcccacg ggtcgaattg cgcttccctgatgagaccga 4400 aaggtcgaaa gtcgaaagac tcggaagcga aagcttggtg atgcattcga4450 tatcgtttaa acgcccgggc gcggccgcgg cgcctccagt cgacgaaagt 4500cggtctgccg aaaggcactg atgagtccga aaggacgaaa ccgacttgct 4550 agataactgatcataatcag ccataccaca tttgtagagg ttttacttgc 4600 tttaaaaaac ctcccacacctccccctgaa cctgaaacat aaaatgaatg 4650 caattgttgt tgttaacttg tttattgcagcttataatgg ttacaaataa 4700 agcaatagca tcacaaattt cacaaataaa gcatttttttcactgcattc 4750 tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta4800 agcgttaatc atgcggccca tgaccaaaat cccttaacgt gagttttcgt 4850tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 4900 cctttttttctgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 4950 accagcggtg gtttgtttgccggatcaaga gctaccaact ctttttccga 5000 aggtaactgg cttcagcaga gcgcagataccaaatactgt ccttctagtg 5050 tagccgtagt taggccacca cttcaagaac tctgtagcaccgcctacata 5100 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt5150 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 5200cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 5250 gacctacaccgaactgagat acctacagcg tgagcattga gaaagcgcca 5300 cgcttcccga agggagaaaggcggacaggt atccggtaag cggcagggtc 5350 ggaacaggag agcgcacgag ggagcttccagggggaaacg cctggtatct 5400 ttatagtcct gtcgggtttc gccacctctg acttgagcgtcgatttttgt 5450 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc5500 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 5550tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 5600 ctgataccgctcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 5650 gaggaagc 5658 18 178DNA Artificial Sequence Multi-cloning sequence flanked by two cis-actingribozymes (CAR′a). 18 gagctcgctt ccctgatgag tccgaaagga cgaaagtcgaaagactcgga 50 agcgaaagct tggtgatgca ttcgatatcg tttaaacgcc cgggcgcggc 100cgcggcgcct ccagtcgacg aaagtcggtc tgccgaaagg cactgatgag 150 tccgaaaggacgaaaccgac ttggtacc 178 19 14 DNA herpes simplex virus 19 cgacgccgcccgcc 14 20 13 DNA herpes simplex virus 20 cugcgcgcgu ggc 13 21 14 DNAherpes simplex virus 21 gcgccugcgc gggg 14 22 14 DNA herpes simplexvirus 22 cgccgccgac gcgc 14 23 13 DNA herpes simplex virus 23 cccccuccccgcg 13 24 14 DNA herpes simplex virus 24 guggccgugu cgcg 14 25 14 DNAherpes simplex virus 25 gccacacggc ggcg 14 26 14 DNA herpes simplexvirus 26 cgccgcgcgg ugcg 14 27 14 DNA herpes simplex virus 27 cgccgcgcggugcg 14 28 15 DNA herpes simplex virus 28 cccccugcgc gccuc 15 29 15 DNAherpes simplex virus 29 gguggugcug uacuc 15 30 14 DNA herpes simplexvirus 30 gggcccgcgg uguc 14 31 14 DNA herpes simplex virus 31 ccuggcgugcgagc 14 32 21 DNA herpes simplex virus 32 ggggaccacc gacgccaugg c 21 3314 DNA herpes simplex virus 33 cguggcgcug gggc 14 34 14 DNA herpessimplex virus 34 cgggauucgc uggg 14 35 15 DNA Artificial SequencePortion of a multiple cloning site for use in making deletion libraries.35 ggtgatgcat tcgat 15 36 38 DNA Artificial Sequence Portion of amultiple cloning site for use in making deletion libraries. 36atcgtttaaa cgcccgggcg cggccgcggc gcctccag 38 37 10 DNA ArtificialSequence Portion of a multiple cloning site for use in making deletionlibraries. 37 ctggaggcgc 10 38 22 DNA Artificial Sequence misc_feature1..16 Portion of an intermediate in the making of a deletion library,including a portion of a multiple cloning site. 38 nnnnnnnnnn nnnnnnctccag 22 39 25 DNA Artificial Sequence misc_feature 6..19 14 bp variablesequence fragment of a deletion library including flanking portions ofmultiple cloning site. 39 ggtgannnnn nnnnnnnnnc tccag 25 40 35 RNAArtificial Sequence misc_feature 1..7 and 31..35 A hammerhead ribozymecomprising a catalytic core flanked by variable recognition domains. 40nnnnnnncug augaggucgc gagaccgaaa nnnnn 35 41 14 RNA Artificial Sequencemisc_feature 1..5 and 8..14 A target substrate comprising variablesequence regions flanking a cleavage site. 41 nnnnnuhnnn nnnn 14 42 12DNA Artificial Sequence misc_feature 6..12 A portion of an antisenselibrary including an HphI site. 42 ggtgannnnn nn 12 43 12 DNA ArtificialSequence misc_feature 1..6 A portion of an antisense library including aBpmI site. 43 nnnnnnctcc ag 12 44 15 DNA Artificial Sequence A sequenceflanking a chloramphenicol (CAT) gene and containing an NruI site. 44ctgatgaggt cgcga 15 45 14 DNA Artificial Sequence A sequence flanking achloramphenicol (CAT) gene and containing an NruI site. 45 tcgcgagagccgaa 14 46 27 DNA Artificial Sequence misc_feature 6..12 Sequenceflanking a chloramphenicol (CAT) gene after insertion into the antisenselibrary. 46 ggtgannnnn nnctgatgag gtcgcga 27 47 25 DNA ArtificialSequence misc_feature 14..19 Sequence flanking the chloramphenicol (CAT)gene after insertion into the antisense library. 47 tcgcgagaccgaannnnnnc tccag 25 48 46 DNA Artificial Sequence misc_feature 6..12 and35..40 Hammerhead ribozyme library with flanking sequences. 48ggtgannnnn nnctgatgag gtcgcgagac cgaannnnnn ctccag 46 49 20 DNAArtificial Sequence misc_feature 1..14 Deletion fragment in a deletionfragment library, including a portion of a multiple cloning site. 49nnnnnnnnnn nnnnctccag 20 50 53 DNA Artificial Sequence Multiple cloningsite for use in making deletion libraries. 50 tgcattcgat atcgtttaaacgcccgggcg cggccgcggc 40 gcctccagtc gac 53

We claim:
 1. A method for generating an oligonucleotide library targetedto a selected RNA transcript comprising: (a) preparing a double strandedcDNA, comprising a first end, a second end, and a central site thereof,from the selected RNA transcript and cloning the cDNA in a cloningvector comprising a promoter configured such that an antisensetranscript of the cDNA is synthesized upon transcription mediated by thepromoter, resulting in a cloned cDNA; (b) creating a plurality ofdeletion derivatives of said cloned cDNA by exonuclease resectionthereof, wherein each of said plurality of deletion derivatives has adeletion extending from said first end towards the central site of thecloned cDNA such that the plurality of deletion derivatives comprises adeletion library comprising deletions that extend serially into thecDNA; and (c) reducing the size of the cDNA contained in the deletionlibrary to a preselected size by removing a portion of the cDNA from thesecond end thereof to result in a fragment library; (d) inserting acatalytic core into the central site of the cDNA in the fragmentlibrary, thereby obtaining the oligonucleotide library.
 2. The method ofclaim 1 wherein said cloning vector comprises a multi-cloning sequencerepresented by SEQ ID NO:1.
 3. The method of claim 1 wherein saidplurality of deletion derivatives is created with exonuclease IIIresection of the cloned cDNA.
 4. The method of claim 1 wherein saidreducing the size of the cDNA contained in the deletion library to apreselected size comprises digesting the deletion library with a typeIIS restriction endonuclease.
 5. The method of claim 1 wherein saidinserting a catalytic core into the central site of the cDNA in thefragment library comprises digesting the fragment library with a typeIIs restriction endonuclease, thereby creating said central site, andligating the catalytic core at the central site.
 6. The method of claim1 wherein said catalytic core comprises a ribozyme catalytic core. 7.The method of claim 1 wherein said catalytic core is a hammerheadribozyme catalytic core.
 8. A multi-cloning sequence represented by SEQID NO:1.